Limits...
HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Bottom Line: HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp).This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin.In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Show MeSH
Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO2-induced methaemoglobin (B).The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in Fig. 1, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO2-treated oxyhaemoglobin, respectively. HRgpA and NaNO2 concentrations were 0.4 µM and 64 µM, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040768&req=5

pone-0017182-g006: Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO2-induced methaemoglobin (B).The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in Fig. 1, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO2-treated oxyhaemoglobin, respectively. HRgpA and NaNO2 concentrations were 0.4 µM and 64 µM, respectively.

Mentions: The facile transfer of iron(III) protoporphyrin IX from methaemoglobin to HmuY was further corroborated by assessment of the relative amount of the HmuY-ferrihaem complex formed from either HRgpA- or NaNO2-induced methaemoglobin. To this end, difference spectra were derived by subtracting the control spectra from the tests at each time interval (Fig. 6A and B) which showed that the amount of HmuY-haem complex formed was greater from methaemoglobin proteolytically induced by HRgpA compared to that produced by NaNO2 treatment.


HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.

Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, Olczak T - PLoS ONE (2011)

Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO2-induced methaemoglobin (B).The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in Fig. 1, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO2-treated oxyhaemoglobin, respectively. HRgpA and NaNO2 concentrations were 0.4 µM and 64 µM, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040768&req=5

pone-0017182-g006: Difference spectra showing HmuY-haem complex formation from HRgpA-methaemoglobin (A) and NaNO2-induced methaemoglobin (B).The difference spectra in the visible region show the appearance of the 525 nm and 558 nm bands attributable to formation of the HmuY-ferrihaem complex. Note that the relative extinctions at 527 nm and 558 nm are reversed compared to those in the ferrihaem-HmuY complex shown in Fig. 1, and occur as a result of subtraction of the spectra. Starting concentrations of haemoglobin were 16 µM (haemoglobin subunit basis) and contained 77 and 87% methaemoglobin for the HRgpA- and NaNO2-treated oxyhaemoglobin, respectively. HRgpA and NaNO2 concentrations were 0.4 µM and 64 µM, respectively.
Mentions: The facile transfer of iron(III) protoporphyrin IX from methaemoglobin to HmuY was further corroborated by assessment of the relative amount of the HmuY-ferrihaem complex formed from either HRgpA- or NaNO2-induced methaemoglobin. To this end, difference spectra were derived by subtracting the control spectra from the tests at each time interval (Fig. 6A and B) which showed that the amount of HmuY-haem complex formed was greater from methaemoglobin proteolytically induced by HRgpA compared to that produced by NaNO2 treatment.

Bottom Line: HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp).This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin.In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, [corrected] University of Liverpool, Liverpool, United Kingdom. josmall@liv.ac.uk

ABSTRACT
Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Show MeSH